High-throughput amplicon sequencing has transformed microsatellite (STR) genotyping by overcoming many of the limitations of fragment-length analysis, enabling more accurate, cost-effective, and standardized genotyping. Yet, protocols specifically designed for high-throughput sequencing (HTS)-based STR genotyping from low-template and degraded DNA remain scarce, despite the prevalence of these challenging sample types in ecological and conservation contexts. We present a methodology for the de novo development of robust STR multiplex panels together with a laboratory protocol for efficient and reliable STR genotyping by sequencing with low quantity and quality DNA samples. The protocol comprises (i) an automated bioinformatic pipeline to design large sets of short tetranucleotide markers optimized for multiplex amplicon sequencing of degraded and low-template DNA; (ii) guidelines for efficient in vitro optimization of multiplex amplification using directly low quantity/quality template DNA; and (iii) a library preparation procedure that improves detection of low-level allele signal while enabling quality assessment of STR amplicon sequencing under limiting DNA conditions. We demonstrate the approach by developing and validating STR panels for non-invasive genotyping of three large carnivore species: a 44-plex for the grey wolf (Canis lupus), a 41-plex for the Eurasian lynx (Lynx lynx), and a 30-plex for the brown bear (Ursus arctos). Multiplex performance was high, with ≥91% of samples successfully genotyped at ≥50% of loci (allele size range 28-110 bp across panels) and correctly assigned to known individuals, negligible levels of noise in the controls, and high discriminatory power (PIDsibs ≤2.4 x 1e-12), also owing to sequence variation among same-length alleles at 15-50% of loci. The approach is broadly applicable to animal and plant species, a wide range of sample types, and large-scale analysis such as genetic monitoring. Our study reinforces the value of STR amplicon sequencing for ecological and conservation applications while highlighting the importance of marker design and laboratory workflows tailored to HTS-based genotyping for accurate and efficient implementation.

Ctenomys de Blainville 1826 ranks among the 10 most species-rich extant mammal genera. However, the taxonomic history of Ctenomys brasiliensis de Blainville 1826-the type species of the genus-has long been obscured by limited information on the collection data of its type material, further complicated by an uncertain geographic origin. In this study, we employed ancient DNA techniques to sequence the complete mitogenome of the extant syntype specimen and conducted an extensive historical investigation to reconcile the original locality names with current geographic designations in South America. Our results unequivocally demonstrate that the type specimen corresponds to the species currently recognized as Ctenomys minutus Nehring 1887. This finding resolves a century-old controversy regarding the provenance of the C. brasiliensis syntype, refuting previous hypotheses that proposed southeastern Brazil or Uruguay as its collection site. Instead, evidence points to a third, previously overlooked location in southernmost Brazil. Earlier studies failed to identify this location due to confusion arising from outdated geographic nomenclature and labeling inaccuracies-issues addressed here through an integrative mitogenomic and historical approach. Complementary quantitative morphological analyses reinforce these conclusions, revealing a close affinity between C. brasiliensis and C. minutus within the same species group. Accordingly, we validate C. brasiliensis and propose C. minutus as its junior synonym. Our study highlights the critical role of robust DNA analyses combined with historical data in clarifying the identity and geographic origin of type specimens, particularly in taxa like Ctenomys, characterized by phenotypic similarity and specimens collected centuries ago.LSID:Version of Record, first published online January 11, 2019, with fixed content and layout in compliance with Art. 8.1.3.2 ICZN.Nomenclature statement - A life science identifier (LSID) number was obtained for this publication: urn:lsid:zoobank.org:pub:6CCE7CD0-2722-4041-9659-1CF50407775D. Ancient DNA from a nearly 200-year-old specimen has finally resolved a long-standing taxonomic mystery surrounding the tuco-tuco genus Ctenomys. Our study reveals that the type specimen, previously thought to be from central Brazil or Uruguay, is actually the species currently known as Ctenomys minutus, occurring in southern Brazil. This finding clarifies its true geographic origin and leads to the reclassification of C. minutus as a synonym, highlighting the critical role of ancient DNA in modern taxonomy. Ctenomys de Blainville 1826 est & aacute; entre os 10 g & ecirc;neros de mam & iacute;feros com maior riqueza de esp & eacute;cies. No entanto, a hist & oacute;ria taxon & ocirc;mica de Ctenomys brasiliensis de Blainville 1826-a esp & eacute;cie-tipo do g & ecirc;nero-tem sido longamente obscurecida pela escassez de informa & ccedil;& otilde;es sobre os dados de coleta de seu material tipo, agravada por uma origem geogr & aacute;fica incerta. Neste estudo, utilizamos t & eacute;cnicas de DNA antigo para sequenciar o mitogenoma completo do esp & eacute;cime s & iacute;ntipo remanescente e conduzimos uma investiga & ccedil;& atilde;o hist & oacute;rica detalhada para reconciliar os nomes das localidades originais com as designa & ccedil;& otilde;es geogr & aacute;ficas atuais na Am & eacute;rica do Sul. Nossos resultados demonstram inequivocamente que o esp & eacute;cime tipo corresponde & agrave; esp & eacute;cie atualmente reconhecida como Ctenomys minutus Nehring 1887. Essa descoberta resolve uma controv & eacute;rsia centen & aacute;ria sobre a proced & ecirc;ncia do s & iacute;ntipo de C. brasiliensis, refutando hip & oacute;teses anteriores que propunham o sudeste do Brasil ou o Uruguai como local de coleta. Em vez disso, as evid & ecirc;ncias apontam para um terceiro local, anteriormente negligenciado, no extremo sul do Brasil. Estudos anteriores n & atilde;o identificaram essa localidade devido & agrave; confus & atilde;o provocada por nomenclaturas geogr & aacute;ficas obsoletas e imprecis & otilde;es nas etiquetas de museu-quest & otilde;es aqui enfrentadas por meio de uma abordagem integrativa, combinando dados mitogen & ocirc;micos e hist & oacute;ricos. An & aacute;lises morfol & oacute;gicas quantitativas complementares refor & ccedil;am essas conclus & otilde;es, revelando uma afinidade estreita entre C. brasiliensis e C. minutus dentro do mesmo grupo de esp & eacute;cies. Assim, validamos C. brasiliensis e propomos C. minutus como seu sin & ocirc;nimo j & uacute;nior. Nosso estudo destaca o papel fundamental de an & aacute;lises gen & eacute;ticas robustas aliadas a dados hist & oacute;ricos para elucidar a identidade e a origem geogr & aacute;fica de esp & eacute;cimes-tipo, especialmente em tax & oacute;ns como Ctenomys, caracterizados por alta similaridade fenot & iacute;pica e por possu & iacute;rem esp & eacute;cimes coletados h & aacute; s & eacute;culos.

A large number of marker typologies have been developed for the metabarcoding of environmental DNA (eDNA). Generalist markers have been advocated for their ability to amplify many taxa simultaneously and produce exhaustive biodiversity assessments, while more specific markers have been proposed for their higher ability to detect and discriminate taxa within a more restricted group. Quantitative comparisons between generalist and specific markers are needed to assess their relative efficiency depending on study targets. Here we compared the performance of one generalist (Euka02, amplifying all eukaryotic groups), one intermediate (Arth02, amplifying arthropods), and two specific markers (Coll01 and Inse01, amplifying springtails and insects, respectively) for the assessment of springtails and insects diversity using eDNA metabarcoding. The four markers were used to analyze eDNA extracted from > 1200 soil samples collected in recently deglaciated terrains. We then assessed whether the different markers were able to detect community responses to key environmental drivers (soil temperature, time since glacier retreat, plant productivity, and topographic wetness). The two specific markers detected more taxa of both springtails and insects than the generalist markers; still the taxonomic richness and community dissimilarity were well correlated between generalist and specific markers. There was good overlap between the taxa identified by the specific markers and those identified by the generalist ones, but the specific markers often detected the taxa identified by the generalist markers, plus several additional ones. Both generalist and specific markers detected the impact of key environmental drivers on arthropods; still the specific markers showed the strongest power to detect relationships, and the most generalist marker was unable to identify the weaker relationships. Generalist markers efficiently provide an overall view of soil biodiversity; nevertheless complementing them with specific markers allows more detailed biodiversity measures, and provides a clearer picture of its response to environmental stressors.

Exploring the biodiversity hidden in tropical rainforests canopies represents a major frontier in biodiversity research yet remains challenging. Environmental DNA (eDNA) can revolutionize this field as it did already in various ecosystems. Here, we test the hypothesis that eDNA contained in canopy throughfall could be used to monitor this elusive diversity and detect anthropogenic disturbance. Using custom-made, low-cost rain collectors, we sampled rainwash eDNA in a mature Amazonian forest and a nearby tree plantation. We successfully detected eDNA from tropical woody and epiphyte plants, vertebrates (mammals, birds, and amphibians), and insects (e.g., mosquitoes, ants, and beetles). The taxonomic composition and diversity reflected disturbance, with significantly lower diversity in the plantation. Crucially, rainwash eDNA integrated biodiversity over a 10-day period in passive collectors and provided a local signature. This approach has thus potential for establishing a cost-effective monitoring system for tropical moist forest canopies, applicable in impact assessments and sustainable management.

Tropical rainforests are vital for global biogeochemical cycles and human well-being and shelter a tremendous, unique, yet underexplored reservoir of biodiversity. With the increasing pressures they face, including deforestation, biological invasions, and climate change, improving methods to monitor their biodiversity is now a pressing societal demand. In recent years, the amplification and sequencing of taxonomically-informative DNA fragments from environmental samples (i.e. eDNA) has revolutionised biomonitoring. Soil, invertebrates bulk, marine and freshwater, or even air, are commonly sampled environmental matrices for such purposes, but present several caveats for the sampling of terrestrial aboveground biodiversity. Here, we explore the potential of DNA contained in rainwash water collected below the forest canopy. We show that it contains not only DNA from invertebrates, but also from the many plants and vertebrates that thrive in the forest canopy. By sampling rainwash eDNA in two 1ha-plots from a tree plantation, and an old-growth Amazonian forest, we detected 170 plant taxa, mainly trees, 72 vertebrate taxa mainly consisting of mammals, birds, and amphibians, and 313 insect taxa including mosquitoes, ants, beetles, etc. The taxonomic composition retrieved in these two plots reflected their different disturbance status. Rainwash eDNA can be efficiently collected passively and persists over ten days while providing a local picture of the diversity. These criteria are compatible with field and environmental management constraints, making the approach promising for an efficient, cost-effective large-scale biomonitoring of tropical rainforest, and more generally all forest canopies. ### Competing Interest Statement The authors have declared no competing interest.

AimIt has been proposed that species diversity (SD) and genetic diversity (GD) co-vary across natural communities because both are shaped by processes such as immigration and drift. However, empirical reports are contradictory, and multispecies studies are rare. Here we test the hypothesis that the two diversity measures do not correlate in systems with high levels of immigration stochasticity and little GD caused by strong genetic drift.LocationTropical alpine habitats on six of the highest mountains in eastern Africa.TaxonVascular plants.MethodsWe sampled 375 taxa in 75 plots in five habitat types, recorded ecological variables, and genotyped 1793 plants representing 20 species/species complexes.ResultsWe confirmed that intrapopulation GD was exceptionally low in this system and found that most Species-Genetic Diversity Correlations (SGDCs) were weak and insignificant. Whereas SD was correlated with several environmental variables, GD was only correlated with mountain identity (geographical location) and mountain age.Main ConclusionsOur findings support the hypothesis that SGDCs are lacking in habitat island systems such as the tropical alpine region in Africa, which is characterised by frequent population size fluctuations, extinctions, and colonisations via long-distance dispersal (LDD) during the glacial cycles. The stochastic nature of LDD combined with strong genetic drift and founder effects is likely to cause low GD and lack of SGDCs, implying that SD cannot be used as a proxy for GD in conservation management of such systems.

Across the northern hemisphere, ungulates are expanding in range and abundance, forming novel communities in increasingly human-modified landscapes. These shifts drive new interactions over available food resources, but patterns of resource use and partitioning in Europe's multi-species systems remain poorly understood. This study examined seasonal diets and resource partitioning in diverse cervid communities (moose, roe deer, red deer, and fallow deer) across two Swedish landscapes (coastal-boreal and boreo-nemoral) differing in deer density and land use. Based on their foraging strategies, we expected (Hypothesis 1) diet richness and dietary niche width to be greater in intermediate feeders (red and fallow deer) than in browsers (moose and roe deer), (Hypothesis 2) trophic partitioning between browsers and intermediate feeders to be driven mainly by graminoid use, and (Hypothesis 3) intra- and interspecific overlap to vary with season, deer density, habitat diversity, and proportion of arable land. DNA metabarcoding of 2568 fecal samples showed that deer consumed plants from over 70 families, though diets were typically dominated by fewer than 10. Vaccinium shrubs were key forages year-round, while birch and willow dominated during the growing season. Moose consumed large amounts of pine in spring and winter (> 50% in the boreo-nemoral, 35%-40% in the coastal-boreal landscape), with less during summer-autumn (~15%). Forbs were important for smaller deer, especially in spring and summer-autumn, and more heavily used in winter in the boreo-nemoral landscape, likely due to supplementary feeding with human-provided food like hay or silage. Spruce use was low overall (< 5%), with fallow deer showing the highest intake. Consistent with Hypothesis 1, diet richness and niche width increased from moose to fallow deer. In partial support of Hypothesis 2, principal coordinates analysis (PCoA) revealed that graminoids contributed to trophic partitioning, but the pattern was not a strict browser-intermediate feeder divide. Moose consistently separated from the smaller deer due to avoidance of graminoids and reliance on pine and juniper, while roe deer, although a browser, sometimes overlapped with red and fallow deer through greater use of graminoids. During winter in the coastal-boreal landscape, wavy hairgrass (Avenella flexuosa) contributed to the significant separation between browsing roe deer and intermediate-feeding red deer diets, consistent with Hypothesis 2. Diet overlap among smaller deer varied with season and landscape. Intraspecific overlap was the highest in moose and the lowest in fallow deer, declining during summer-autumn across species. Overlap was influenced by deer density, habitat diversity, and arable land, consistent with Hypothesis 3, but effects were species-specific and explained only limited variation. Our results highlight the dietary plasticity of red and fallow deer, which may intensify resource competition with moose and roe deer in multi-species systems, particularly where supplementary feeding is common. These insights support adaptive, multi-species management of deer in northern ecosystems.

Accurate detection and identification of vector-host-parasite systems are key to understanding their evolutionary dynamics and to design effective disease prevention strategies. Traditionally, microscopical and serological techniques were employed to analyse arthropod blood meals for host/parasite detection, but these were limited in taxonomic resolution and only to pre-selected taxa. In recent years, molecular techniques have emerged as a promising alternative, offering enhanced resolution and taxonomic range. While singleplex polymerase chain reaction (PCR) assays were used at first to identify host, vector and parasite components in separate reactions, today multiple primer pairs can be combined in a single reaction, i.e., multiplex, offering substantial time and cost savings. Nonetheless, despite the potential benefits of multiplex PCR, studies quantifying its efficacy compared to singleplex reactions are scarce. In this study, we used partially digested mosquito blood meals within an avian malaria framework to jointly identify the host, vector and parasite using multiplex DNA metabarcoding, and to compare it with separate singleplex PCRs. We aimed to compare the detection probabilities and taxonomic assignments between both approaches. We found both to have similar performances in terms of detection for the host and the vector, but singleplex clearly outperformed multiplex for the parasite component. We suggest adjusting the relative concentrations of the PCR primers used in the multiplex assay could increase the efficiency of multiplex in detecting all the components of the studied multi-species system. Overall, the results show that multiplex DNA metabarcoding can be an effective approach that could be applied to any vector-borne interaction involving blood-feeding arthropods. Our insights from this proof-of-concept study will help improve laboratory procedures for accurate and cost-efficient medical diagnosis of vector-borne diseases, the spread of which is globally exacerbated by current climate change.

Different Savoyard cheeses are granted with PDO (Protected Designation or Origin) and PGI (Protected Geographical Indication) which guarantees consumers compliance with strict specifications. The use of raw milk is known to be crucial for specific flavor development. To unravel the factors influencing microbial ecosystems across cheese making steps, according to the seasonality (winter and summer) and the mode of production (farmhouse and dairy factory ones), gene targeting on bacteria and fungus was used to have a full picture of 3 cheese making technologies, from the raw milk to the end of the ripening. Our results revealed that Savoyard raw milks are a plenteous source of biodiversity together with the brines used during the process, that may support the development of specific features for each cheese. It was shown that rinds and curds have very contrasted ecosystem diversity, composition, and evolution. Ripening stage was selective for some bacterial species, whereas fungus were mainly ubiquitous in dairy samples. All ripening stages are impacted by the type of cheese technologies, with a higher impact on bacterial communities, except for fungal rind communities, for which the technology is the more discriminant. The specific microorganism's abundance for each technology allow to see a real bar-code, with more or less differences regarding bacterial or fungal communities. Bacterial structuration is shaped mainly by matrices, differently regarding technologies while the influence of technology is higher for fungi. Production types showed 10 differential bacterial species, farmhouses showed more ripening taxa, while dairy factory products showing more lactic acid bacteria. Meanwhile, seasonality looks to be a minor element for the comprehension of both microbial ecosystems, but the uniqueness of each dairy plant is a key explicative feature, more for bacteria than for fungus communities.

The worldwide retreat of glaciers is causing a faster than ever increase in ice-free areas that are leading to the emergence of new ecosystems. Understanding the dynamics of these environments is critical to predicting the consequences of climate change on mountains and at high latitudes. Climatic differences between regions of the world could modulate the emergence of biodiversity and functionality after glacier retreat, yet global tests of this hypothesis are lacking. Nematodes are the most abundant soil animals, with keystone roles in ecosystem functioning, but the lack of global-scale studies limits our understanding of how the taxonomic and functional diversity of nematodes changes during the colonization of proglacial landscapes. We used environmental DNA metabarcoding to characterize nematode communities of 48 glacier forelands from five continents. We assessed how different facets of biodiversity change with the age of deglaciated terrains and tested the hypothesis that colonization patterns are different across forelands with different climatic conditions. Nematodes colonized ice-free areas almost immediately. Both taxonomic and functional richness quickly increased over time, but the increase in nematode diversity was modulated by climate, so that colonization started earlier in forelands with mild summer temperatures. Colder forelands initially hosted poor communities, but the colonization rate then accelerated, eventually leveling biodiversity differences between climatic regimes in the long term. Immediately after glacier retreat, communities were dominated by colonizer taxa with short generation time and r-ecological strategy but community composition shifted through time, with increased frequency of more persister taxa with K-ecological strategy. These changes mostly occurred through the addition of new traits instead of their replacement during succession. The effects of local climate on nematode colonization led to heterogeneous but predictable patterns around the world that likely affect soil communities and overall ecosystem development.

Ctenomys [Blainville 1826][1] ranks among the top ten most diverse mammal genera in terms of species richness. However, the taxonomic history of Ctenomys brasiliensis [Blainville,1826][1], the corresponding type species, has long been obscured by a dearth of information regarding the collection data of the type material, compounded by an elusive geographic origin. Here, employing ancient DNA methodology, we sequenced the complete mitogenome of the remaining type specimen and conducted an extensive historical investigation to correlate originally described locality names with present-day locales in South America. Our analysis unequivocally confirms that the type specimen corresponds to the species currently designated as Ctenomys minutus [Nehring, 1887][2]. This resolution lays to rest a century-old debate surrounding the provenance of the type specimen, rejecting prior hypotheses that placed its collection site in southeastern Brazil or Uruguay. Instead, our evidence suggests it was likely obtained from a third location in southernmost Brazil. Previous analyses overlooked this new location due to confusion surrounding geographic nomenclature and labeling errors, issues rectified by our combined mitogenomic and historical approach. Furthermore, quantitative morphological analyses boost our findings, demonstrating a closer affinity between C. brasiliensis and C. minutus within the same species group. Accordingly, we validate C. brasiliensis and propose C. minutus as its junior synonym. Our study underscores the importance of robust DNA analyses in confirming the identity and geographic origins of type specimens, especially for Ctenomys species with similar phenotypes, and specimens collected centuries ago. ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-7 [2]: #ref-36

The global retreat of glaciers is dramatically altering mountain and high-latitude landscapes, with new ecosystems developing from apparently barren substrates1-4. The study of these emerging ecosystems is critical to understanding how climate change interacts with microhabitat and biotic communities and determines the future of ice-free terrains1,5. Here, using a comprehensive characterization of ecosystems (soil properties, microclimate, productivity and biodiversity by environmental DNA metabarcoding6) across 46 proglacial landscapes worldwide, we found that all the environmental properties change with time since glaciers retreated, and that temperature modulates the accumulation of soil nutrients. The richness of bacteria, fungi, plants and animals increases with time since deglaciation, but their temporal patterns differ. Microorganisms colonized most rapidly in the first decades after glacier retreat, whereas most macroorganisms took longer. Increased habitat suitability, growing complexity of biotic interactions and temporal colonization all contribute to the increase in biodiversity over time. These processes also modify community composition for all the groups of organisms. Plant communities show positive links with all other biodiversity components and have a key role in ecosystem development. These unifying patterns provide new insights into the early dynamics of deglaciated terrains and highlight the need for integrated surveillance of their multiple environmental properties5.

Sedimentary ancient DNA (sedaDNA) has rarely been used to obtain population-level data due to either a lack of taxonomic resolution for the molecular method used, limitations in the reference material or inefficient methods. Here, we present the potential of multiplexing different PCR primers to retrieve population-level genetic data from sedaDNA samples. Vaccinium uliginosum (Ericaceae) is a widespread species with a circumpolar distribution and three lineages in present-day populations. We searched 18 plastid genomes for intraspecific variable regions and developed 61 primer sets to target these. Initial multiplex PCR testing resulted in a final set of 38 primer sets. These primer sets were used to analyse 20 lake sedaDNA samples (11,200 cal. yr BP to present) from five different localities in northern Norway, the Alps and the Polar Urals. All known V. uliginosum lineages in these regions and all primer sets could be recovered from the sedaDNA data. For each sample on average 28.1 primer sets, representing 34.15 sequence variants, were recovered. All sediment samples were dominated by a single lineage, except three Alpine samples which had co-occurrence of two different lineages. Furthermore, lineage turnover was observed in the Alps and northern Norway, suggesting that present-day phylogeographical studies may overlook past genetic patterns. Multiplexing primer is a promising tool for generating population-level genetic information from sedaDNA. The relatively simple method, combined with high sensitivity, provides a scalable method which will allow researchers to track populations through time and space using environmental DNA.

Several sympatric deer species that utilize the same, limited resources during the lean season may compete for food resources and thus strongly affect forest regeneration and increase wildlife-forestry conflicts. Using fecal DNA metabarcoding, we studied winter diet compositions, food preferences, and dietary niche partitioning among moose, roe deer, and red deer in NE Poland during harsh (snowy) and mild winters to test the competition avoidance hypothesis. In coniferous and mixed forests, these three species exhibited browsing strategy, as their winter diets were dominated by a high proportion of woody browse (>99% of plant DNA sequences in moose, >90% in roe deer, and >79% in red deer fecal samples) and contained almost no grasses. Dietary richness per fecal sample was the lowest for moose (9.8), higher for roe deer (19.6), and the highest for red deer (26.3), as it was also composed of different forbs and ferns. The studied cervids were selective and strongly preferred Scots pine, Maleae (incl. rowan tree), Salix sp. and the ericaceous dwarf shrubs, and avoided Norway spruce, silver birch, oak, bramble, and alder buckthorn. During mild winters, increased browsing by roe deer and red deer on the ericaceous dwarf shrubs resulted in the overlap of their diets. When the snow cover depth exceeded 10 cm, these two deer species effectively partitioned their diets, in line with the hypothesis of competition avoidance for food resources. During harsh winter, however, the diets of roe deer and red deer overlapped to a larger extent with the moose 'conifer diet', resulting in possible competition for food. In effect, the joint browsing of native European deer species on young coniferous trees during harsh winter was larger than that during mild winters. The same could be the case in managed forests where available forage is usually scarce. These findings may have profound consequences for future forest and ungulate management under ongoing climate change.

1. The current biodiversity crisis demands a shift from single-species to multispecies approaches in conservation, particularly for rare and endangered species. However, this transition requires tools optimised for multispecies research, which are currently limited. 2. Recent advances in high-throughput sequencing (HTS) technologies and bioinformatics have enabled efficient and robust acquisition of genetic data. Amplicon sequencing approaches, in particular, have demonstrated potential for enhancing non-invasive genetic studies of endangered species, but their application has been mostly limited to single species. 3. To enable multispecies genetic research, we introduce a cost-effective and robust HTS-based amplicon sequencing approach for genotyping multiple species simultaneously, designed for population monitoring, including individual identification and ascertaining patterns of population structure. 4. We developed Feliplex, a multiplex panel of 85 co-amplifying tetranucleotide microsatellite markers for cross-genotyping Felidae species, to demonstrate the utility of our approach. Feliplex was validated on known samples from nine Indian felid species across the genera Panthera, Prionailurus, and Felis. We applied it to invasive (blood and tissue) and non-invasive (hair and faeces) DNA extracts from 173 wild individuals obtaining respectively 70% and 56% multilocus genotyping success rates. The panel accurately identified known population clusters in tigers (Panthera tigris, n=19) and revealed hitherto unknown genetic structure in fishing cats (Prionailurus viverrinus, n=40). 5. Wide applicability of Feliplex across Felidae allows reliable multispecies genotyping from low-quality/quantity samples, while supporting cost-effective genetic studies and conservation monitoring of lesser-known species like small cats. Our approach has a broad applicability and can be adapted to develop similar multispecies panels for closely related species groups. ### Competing Interest Statement The authors have declared no competing interest.

The mechanisms underlying plant succession remain highly debated. Due to the local scope of most studies, we lack a global quantification of the relative importance of species addition ‘versus’ replacement. We assessed the role of these processes in the variation (β-diversity) of plant communities colonizing the forelands of 46 retreating glaciers worldwide, using both environmental DNA and traditional surveys. Our findings indicate that addition and replacement concur in determining community changes in deglaciated sites, but their relative importance varied over time. Taxa addition dominated immediately after glacier retreat, as expected in harsh environments, while replacement became more important for late-successional communities. These changes were aligned with total β-diversity changes, which were more pronounced between early-successional communities than between late-successional communities (>50 yr since glacier retreat). Despite the complexity of community assembly during plant succession, the observed global pattern suggests a generalized shift from the dominance of facilitation and/or stochastic processes in early-successional communities to a predominance of competition later on.

Summary The development of terrestrial ecosystems depends greatly on plant mutualists such as mycorrhizal fungi. The global retreat of glaciers exposes nutrient‐poor substrates in extreme environments and provides a unique opportunity to study early successions of mycorrhizal fungi by assessing their dynamics and drivers. We combined environmental DNA metabarcoding and measurements of local conditions to assess the succession of mycorrhizal communities during soil development in 46 glacier forelands around the globe, testing whether dynamics and drivers differ between mycorrhizal types. Mycorrhizal fungi colonized deglaciated areas very quickly (< 10 yr), with arbuscular mycorrhizal fungi tending to become more diverse through time compared to ectomycorrhizal fungi. Both alpha‐ and beta‐diversity of arbuscular mycorrhizal fungi were significantly related to time since glacier retreat and plant communities, while microclimate and primary productivity were more important for ectomycorrhizal fungi. The richness and composition of mycorrhizal communities were also significantly explained by soil chemistry, highlighting the importance of microhabitat for community dynamics. The acceleration of ice melt and the modifications of microclimate forecasted by climate change scenarios are expected to impact the diversity of mycorrhizal partners. These changes could alter the interactions underlying biotic colonization and belowground–aboveground linkages, with multifaceted impacts on soil development and associated ecological processes.

Many plant species develop fruits to attract animals that will eat them and then disperse the seeds. However, there are many plant species, whose seeds are dispersed endozoochorically, but their fruits are not particularly attractive to animals. The “Foliage is the fruit” (FF) hypothesis proposes that entire biomass of the plant exists to encourage herbivores to eat it, in order to enhance seed intake and dispersal (Janzen, 1984). We tested the FF hypothesis by combining the results from the greenhouse seedling emergence method (GR) and DNA-metabarcoding of plant remnants in faeces of European moose (Alces alces L.). We processed 665 samples by the GR and 429 by the MB method, hypothesizing that if the safe passage of seeds through the gut of a large herbivore is the result of an evolutionary adaptation to endozoochoric dispersal, then the species composition of plants revealed by the two methods should largely overlap and the abundance of seedlings revealed by the GR method should be positively correlated in time with the read abundance of DNA of the same species. The large discrepancy between the lists of species detected by DNA metabarcoding and the GR method argues against the FF hypothesis. However, in the case of Urtica dioica, Lysimachia vulgaris and Lythrum salicaria, some clues of evolutionary adaptation to endozoochoric dispersal were revealed for: 1) their foliage is attractive to herbivores; 2) seeds are small, rounded in shape, yielded in large numbers and pass safely through the herbivore's gut; 3) the abundance of seeds (seedlings) was significantly and strongly influenced by the abundance of the plant biomass (DNA reads) in dung samples; 4) peaks in seed abundance and biomass consumption coincided in time. However, it should be considered that moose's diet is mostly composed of woody browse, which makes this animal not an optimal model for testing the Janzen's hypothesis and studies on typical grazers are needed in this respect.

The question of how local adaptation takes place remains a fundamental question in evolutionary biology. The variation of allele frequencies in genes under selection over environmental gradients remains mainly theoretical and its empirical assessment would help understanding how adaptation happens over environmental clines. To bring new insights to this issue we set up a broad framework which aimed to compare the adaptive trajectories over environmental clines in two domesticated mammal species co-distributed in diversified landscapes. We sequenced the genomes of 160 sheep and 161 goats extensively managed along environmental gradients, including temperature, rainfall, seasonality and altitude, to identify genes and biological processes shaping local adaptation. Allele frequencies at putatively adaptive loci were rarely found to vary gradually along environmental gradients, but rather displayed a discontinuous shift at the extremities of environmental clines. Of the 430 candidate adaptive genes identified, only 6 were orthologous between sheep and goats and those responded differently to environmental pressures, suggesting different putative mechanisms involved in local adaptation in these two closely related species. Interestingly, the genomes of the 2 species were impacted differently by the environment, genes related to signatures of selection were most related to altitude, slope and rainfall seasonality for sheep, and summer temperature and spring rainfall for goats. The diversity of candidate adaptive pathways may result from a high number of biological functions involved in the adaptations to multiple eco-climatic gradients, and a differential role of climatic drivers on the two species, despite their co-distribution along the same environmental gradients. This study describes empirical examples of clinal variation in putatively adaptive alleles with different patterns in allele frequency distributions over continuous environmental gradients, thus showing the diversity of genetic responses in adaptive landscapes and opening new horizons for understanding genomics of adaptation in mammalian species and beyond.

Metabarcoding analyses have recently undergone significant development due to the power of this technique in biodiversity monitoring. However, it is still difficult to draw accurate quantitative conclusions about the ecosystems studied, mainly because of biases inherent in the environmental DNA or introduced during the experimental process. These biases alter the relationship between the amount of DNA observed and the biomass or number of individuals of the species detected. Two of the biases inherent in metabarcoding have been measured: the ratio between total DNA and target DNA concentrations, and the PCR amplification bias. A method for their correction is proposed. All experimental tests were performed on mock alpine plant communities using the marker Sper01 , which is expected to have low amplification bias due to its highly conserved priming sites. Our approach combines standard quantitative PCR techniques (qPCR and digital droplet PCR) with a realistic stochastic model of PCR dynamics that accounts for PCR saturation. The model was used to estimate PCR efficiencies for each species and to infer the true species proportions of the mock communities from the read relative frequencies. The corrections are easy to implement and can be applied to previously generated DNA metabarcoding data. This work demonstrates the relative importance of the two biases considered and is an open door to quantitative metabarcoding data, although many other biases remain to be considered.